For the previous funding period we proposed to develop a small molecule mimetic of the pro-apoptotic protein Smac. The idea was to promote or sensitize cancer cells to apoptosis in situations where they resist executing the pathway.We have succeeded in this goal. Smac is normally a mitochondrial protein and is released into the cytosol only during apoptosis. Once in the cytoplasm, Smac binds to several Inhibitor-of- Apoptosis Proteins (lAPs) and neutralizes their ability to inhibit caspases, a group of intracellular proteases that execute apoptosis. The functional motif in Smac is found at its very N-terminus, with just four residues (AVPI) being sufficient and necessary for Smac like activity in vitro. Based on this information, we designed and characterized a small molecule that performs just as well, if not better, than the native protein in terms of relieving IAP inhibition of caspases. More importantly, our compound is cell permeable and at subnanomolar concentrations synergizes with death inducing cytokines such as tumor necrosis factor alpha (TNF-a) and TNF-related apoptosis-inducing ligand (TRAIL) to facilitate cell death in cancer cell lines that do not respond to these cytokines alone. The compound is not toxic towards non-cancerous cells. We have now evaluated more than 80 cultured human cancer cell lines and found that responses vary from highly sensitive to resistant. This gives us a unique opportunity to further understand signaling in apoptosis. In particular, by learning why certain cancer cells undergo programmed cell death in the presence of a Smac mimetic alone, while others do so only with an added stimulus, or not at all.This proposal outlines detailed biochemical, molecular and cell biological experiments to probe this issue. This research is relevant to public health because it could help develop a new cancer therapy and guide its use in the clinic.